The Effect of the Combined Action of Roscovitine and Paclitaxel on the Apoptotic and Cell Cycle Regulatory Mechanisms in Colon and Anaplastic Thyroid Cancer Cells

Aim. To study the significance of cyclin-dependent kinases (Cdks) in paclitaxel-dependent apoptosis in colon and undifferentiated thyroid cancer cells. Materials and Methods. Experiments were performed on undifferentiated thyroid carcinoma (KTC-2) and colon carcinoma (ARO) cell lines. Cells were treated with paclitaxel (Ptx) and inhibitor of Cdk, roscovitine. Cell survival test and Western blotting were used for characterization of the effects of paclitaxel and roscovitine on cancer cells. Results. It was shown that not c-Jun N-terminal kinase, but cyclin-dependent kinases are responsible for antiapoptotic Bcl-2 phosphorylation. Cdk inhibition enhanced the cytotoxic effects of Ptx at low drug concentrations. There was antagonism between Ptx and roscovitine at higher (25 nM) paclitaxel concentrations. Conclusion. Using of paclitaxel at low (2.5 to 5 nM) concentrations and roscovitine is a promising combination for further preclinical trials for the development of new therapeutic approaches to the treatment of colon and anaplastic thyroid cancer.


Introduction
Compounds that stabilize microtubules (MSA), which include taxanes (paclitaxel, docetaxel), are effective anticancer drugs. It is known that the therapeutic effect of these drugs is associated with cell cycle arrest, followed by initiation of apoptotic processes [1]. However, the exact mechanism that links cell division impairment with apoptosis induced by drugs is still poorly understood.
The mammalian cell cycle is controlled by cyclindependent kinases, whose activity is modulated by several activators and inhibitors [2]. Cdks are serine/threonine kinases that play a key role in regulating both cell cycle and transcription through the phosphorylation of transcription factors and tumor suppressor proteins involved in DNA replication and cell division [2].
The Cdk modulators include a 2, 6, 9-substituted purine analogue, roscovitine (CYC202), which inhibits Cdk activity directly by competing for the ATP-binding sites of Cdk and causing apoptosis within various tumor cells. A study of clinical utility of roscovitine showed its anticancer effects and limited toxicity for humans in clinical trials [3].
The aim of this study was to establish a connection between the effect of paclitaxel on cell cycle and induction of apoptotic processes in colon (CC) and anaplastic thyroid cancer (ATC) cell lines ARO and KTC-2.  (FBS) and 1% penicillin/streptomycin (all reagents from Invitrogen Life Technologies, Paisley, UK) in a 5% CO 2 humidified atmosphere at 37 • C. After 2 d incubation, when the culture reached about 80% confluence, cells were washed twice with PBS (pH 7.4) at 37 • C, and a fresh medium was added to each dish. Cells were incubated for additional 24 h, exposed to the drug(s) as described below, and then collected at different time intervals.

Cell Survival Assay.
Cultures were established in the 96well flat-bottom microtiter plates in RPMI 1640 containing 5% FBS. Cell suspensions (100 μL, ∼1000 cells/well) were added to each well and incubated for 24 h before treatment. Ptx (Wako Chemicals, Osaka, Japan) and roscovitine dissolved in dimethylsulfoxide (DMSO) and the control (DMSO only) were added to each well at varying concentrations, six wells for each concentration. After incubation, a water-soluble tetrazolium salt-based assay (WST) was performed as follows: 11 μL of the cell counting kit solution (CCK-8, Dojin, Osaka, Japan) were added to each well and incubated for 1 h at 37 • C. OD was read at 450 nm in a microplate reader.

Statistical
Analysis. All data were expressed as a mean ± SD or mean ± SE. Differences between groups were assessed for statistical significance using Student's t test. P < 0.05 denoted the presence of a statistically significant difference.

Results and Discussion
It is believed that the basis of the mechanism of apoptosis induction by Ptx in tumor cell is phosphorylation with subsequent degradation of the antiapoptotic protein Bcl-2, which stabilizes the mitochondrial membrane [4,5].
A decrease in this protein amount leads to an imbalance between pro-and antiapoptotic proteins causing a release of mitochondrial cytochrome C and other apoptotic factors followed by caspase-9 activation. The JNK-dependent phosphorylation of Bcl-2 and its further degradation were considered as the most probable event, which mediates the Ptx action [4]. However, experiment with a JNK inhibitor, * * SP600125, showed that inhibition of JNK in ATC cells did not reduce Bcl-2 phosphorylation as expected but even slightly increased it (Figure 1(1)). Since Ptx in ATC cells activates a number of cell cycle regulators [6], a specific inhibitor of Cdk, roscovitine, was used. It was found that inhibition of Cdk caused a nearly complete, qualitative inhibition of Bcl-2 phosphorylation (Figure 1(2)). Thus, Cdk activated by Ptx is directly or indirectly responsible for phosphorylation of antiapoptotic protein Bcl-2. These results are confirmed by data obtained on KB-3 human carcinoma cell line evidenced that Cdk1 phosphorylated Bcl-X L /Bcl-2 and thus attenuated their antiapoptotic function [7].
It should be noted that JNK, and to a lesser extent p38MAPK, also mediates Ptx-induced apoptosis in ATC cells [4] but obviously through parallel mechanisms. Future experiments will show whether there are any interrelationships between these mechanisms and Cdk or Cdk-dependent signaling. Some data point out the possibility of such an interaction [8,9].
One of the roscovitine targets in the cell is Cdk1 (Cdc2) [10,11], which in combination with cyclin B1 promotes the transition of cells from G2 phase to mitosis. It has been shown that in ATC cells Ptx, like other MSA, stopped the transition, caused an arrest of cycle at the G2/M stage [5]. This triggered the activation of a number of cell cycle regulators, which, on the background of a decrease in quantity of Cdk-inhibitors (p27 KIP1 and p21 WAF1 ), stimulated overcoming of the G2/M barrier [6]. Among the most important events, it should be noted the activation of phosphatase Cdc25C (dephosphorylation of ser216), which in turn activates Cdk1 by removing inhibitory phosphate at position 15 tyrosine residues, and a significant increase in Cdk1 cofactor cyclin B1 expression (Figures 1(3)-1(5)). Perhaps an excessive activation of Cdk1, which is observed under the influence of Ptx, is one of the triggers that initiated the process of apoptosis in tumor cells.
Thus, it may be suggested that the initiation of mitochondrial apoptosis in cells of anaplastic thyroid cancer under the action of Ptx is mediated by cyclin-dependent kinases.
However, a study of the effects of combined action of roscovitine and Ptx upon apoptotic mechanisms showed that roscovitine activated caspase-9, caspase-8, and PARP cleavage but attenuated Ptx-dependent activation of caspase-9, PARP cleavage, and especially caspase-8 activation (Figures 2(a) and 2(b)). On the other hand, roscovitine significantly decreased antiapoptotic XIAP level, which is high under Ptx action ( Figure 2).
Cell survival study on ARO cells showed that after 24 h of incubation roscovitine enhanced Ptx cytotoxicity at low drug concentration, and there were no effects at higher Ptx concentrations: 10-25 nM and after 48 h of incubation (Figures 3(a) and 3(b)). In KTC-2 cells roscovitine, after 24 h of incubation, enhanced Ptx cytotoxicity at all studied drug concentrations (Figure 3(c)). After 48 h of incubation, roscovitine increased Ptx cytotoxicity at low (2.5-5 nM) concentrations and did not affect or even inhibited cell death at higher drug concentrations (Figure 3(d)). Certain inhibition of Ptx cytotoxicity at 25 nM by roscovitine probably reflected the reduced activity of caspases under combined effects of Ptx and roscovitine (Figures 2(a) and 2(b)). The difference in roscovitine action on ARO and KTC-2 cell line as well as a higher sensitivity of KTC-2 cells to the drugs may be due to the presence of an active TP53 gene in the latter. It is known that roscovitine can induce activation and stabilization of p53 by suppression of MDM2 expression [12,13].
Thus, roscovitine in ATC and CC cells showed marked proapoptotic effects, which can be explained by phosphorylation of antiapoptotic protein Bcl-2 and inhibition of IAP expression. There is evidence also that roscovitine suppressed antiapoptotic Mcl-1 expression and downregulated FLICE-inhibitory proteins in breast cancer cells [14]. Other data show that roscovitine increases the proapoptotic Bax and decreases antiapoptotic survivin and XIAP expression, resulting in caspase-dependent apoptosis of sarcoma cells [15].
Combined action of micromolar concentrations of roscovitine and low (1-5 nM) concentrations of Ptx may be a promising strategy for further preclinical investigations.